TET3 epigenetically controls feeding and stress response behaviors via AGRP neurons

The TET family of dioxygenases promote DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Hypothalamic agouti-related peptide–expressing (AGRP-expressing) neurons play an essential role in driving feeding, while also modulating nonfeeding behaviors. Besides AGRP, these neurons produce neuropeptide Y (NPY) and the neurotransmitter GABA, which act in concert to stimulate food intake and decrease energy expenditure. Notably, AGRP, NPY, and GABA can also elicit anxiolytic effects. Here, we report that in adult mouse AGRP neurons, CRISPR-mediated genetic ablation of Tet3, not previously known to be involved in central control of appetite and metabolism, induced hyperphagia, obesity, and diabetes, in addition to a reduction of stress-like behaviors. TET3 deficiency activated AGRP neurons, simultaneously upregulated the expression of Agrp, Npy, and the vesicular GABA transporter Slc32a1, and impeded leptin signaling. In particular, we uncovered a dynamic association of TET3 with the Agrp promoter in response to leptin signaling, which induced 5hmC modification that was associated with a chromatin-modifying complex leading to transcription inhibition, and this regulation occurred in both the mouse models and human cells. Our results unmasked TET3 as a critical central regulator of appetite and energy metabolism and revealed its unexpected dual role in the control of feeding and other complex behaviors through AGRP neurons.

in the cage and monitored for the following 24 h. For ChIP studies, Cas9+ mice injected with AAV or AAV-sgTet3 bilaterally into the ARC were fasted overnight for 22 h. On the second day, saline or leptin (5 mg/kg) was administrated intraperitoneally at 10:00. Two hours later, ARCs were isolated for ChIP-qPCR analysis. To examine leptin-induced protein-protein interactions, Cas9+ mice were fasted overnight for 22 h. On the second day, leptin was administrated at 5 mg/kg intraperitoneally at 10:00, and ARCs were isolated 2 h later, followed by co-IP studies.

Stereotaxic injection
Injections were made into the ARC of anesthetized 6-week-old Cas9+ mice, placed in a stereotaxic apparatus (model 902; Kopf Instruments). Viruses (500 nL, 5x10 12 GC/ml per site of injection) were applied into each hemisphere (coordinates: bregma, anterior-posterior: −1.45 mm, dorsal-ventral: −5.8 mm, lateral: +/-0.27 mm) by using an air pressure system (injection time: 5 minutes). After surgery, mice were allowed to recover for 2 weeks before electrophysiological recording. Stereotaxic injection sites were verified by double fluorescence labeling for GFP and mCherry, which could be detected without immunostaining. Mice with "missed" or "partial" hits were excluded from data analyses.

Osmotic pump installation
Three days after bilateral ARC co-injection with AAV-sgTet3 and AAV-h4MDi, a mini-osmotic pump (model 1007D, Alzet) was implanted subcutaneously. The osmotic pump was filled with either sterile saline solution or DREADD agonist compound 21 (C21) dihydrochloride (0.5 mg/kg, HB6124-25mg, Hello Bio). Food intake measurement and ITT were performed at day 5 and day 9 postinjection, respectively. For food intake assays, food pellets were weighed at 10:00 each day for 3 continuous days and an average of three-day food intake was calculated.

Body weight, body composition, and food intake measurement
Mice were singly housed after surgery. Body weight was measured every other week, and body composition was assessed using EchoMRI analysis. Food intake and energy expenditure were measured using an indirect calorimetry chamber (TSE Systems, Germany).

Electrophysiology
Coronal hypothalamic slices containing the ARC were prepared from virus injected mice as previously reported (5). In brief, mice were anesthetized with isoflurane and decapitated. The brain was rapidly removed and immersed in cold (4 ºC) and oxygenated cutting solution containing (in mM): sucrose 220, KCl 2.5, NaH2PO4 1.23, NaHCO3 26, CaCl2 1, MgCl2 6, and glucose 10 (pH 7.3 with NaOH). Coronal slices (300 μm thick) were prepared with a Leica vibratome after the brain was trimmed to a small tissue block containing the hypothalamus. After preparation, slices were maintained at room temperature (23 ºC -25 ºC) in a storage chamber in artificial cerebrospinal fluid (ACSF) (bubbled with 5% CO2 and 95% O2) containing (in mM): NaCl 124, KCl 3, CaCl2 2, MgCl2 2, NaH2PO4 1.23, NaHCO3 26, glucose 10 (pH 7.4 with NaOH) for recovery and storage. After recovery at room temperature for at least 1 hour, slices were transferred to a recording chamber constantly perfused at a rate of 2 mL/min with ACSF containing 2.5 mM glucose at a temperature of 33 ºC for electrophysiological experiments. To identify virus infected AGRP neurons, mCherry and GFP fluorescence were detected using LED illumination (CoolLED pE-300). Whole-cell patch clamp recordings were obtained from AGRP neurons visualized using infrared differential interference contrast (IR-DIC) imaging. Spontaneous membrane and action potentials (MP) were recorded under current clamp as previously reported (6,7). The micropipettes (4-6 MΩ) were made of borosilicate glass (World Precision Instruments) with a micropipette puller (Sutter P-97) and backfilled with a pipette solution containing (in mM): K-gluconate 108, KCl 27, MgCl2 2, HEPES 10, EGTA 1.1, Mg-ATP 2.5, Na2-GTP 0.3, and Na2-phosphocreatine 10, pH 7.3 with KOH. Both input resistance and series resistance were monitored throughout the experiments, and the former was partially compensated. Only recordings with stable series resistance and input resistance were accepted. All data were sampled at 3 kHz, filtered at 3 kHz, and analyzed with an Apple Macintosh computer using AxoGraph X. t test was used to examine the statistical significance of the difference in AP frequency and threshold in the recorded AGRP neurons.

Bobcat339 treatment of mice
Chow-fed C57BL/6J mice at the age of 12 weeks were treated with Bobcat339 (100 mg/kg per day) or vehicle (DMSO) in drinking water for 4 days. ARCs were isolated at 10:00 from ad libitum-fed mice and subjected to immunofluorescence analysis. Bobcat339 was dissolved in DMSO at a concentration of 100 mg/ml and stored at -20 0 C in aliquots. Working solution (1 mg/ml) was freshly prepared every other day by dilution using tap water.

GTT and ITT
Glucose tolerance tests (GTT) were performed following 16 h overnight fasting. Each animal received an intraperitoneal injection of 2 g/kg glucose (Sigma-Aldrich, G5767) in sterile saline. Insulin tolerance tests (ITT) were performed following a 3 h morning-fasting. Each animal received an intraperitoneal injection of 1 U/kg insulin (Novolin R Regular U-100 insulin) in sterile saline. Blood glucose concentrations were measured using Contour next blood glucose meter (Ascensia Diabetes Care) via tail vein bleeding at the indicated time points after injection.

Hydroxymethylated DNA immunoprecipitation coupled with qPCR (hMeDIP-qPCR)
The experiments were carried out using the EpiQuik hMeDIP Kit (P-1038-48, Epigentek) according to the manufacturer's instructions. Briefly, for ARC hMeDIP, freshly isolated ARCs (2 ARCs from one mouse per IP, Figure 4K) were washed twice with 1 ml of cold PBS and homogenized (5-10 strokes) using a disposable pellet pestle (Fisher Scientific, 12-141-368) in 500 μl of Genomic Lysis Buffer. For SH-SY5Y hMeDIP ( Figure 4L), cells seeded in 6-well plates at 1x10 6 cells/well the night before were transfected with NT siRNA or TET3 siRNA under Lept H conditions, and genomic DNAs were isolated at 48 h following transfection using Quick gDNA MicroPrep Kit (D3021, Zymo Research Corporation) and sheared using a sonifier (Branson 150), with a setting of 9 pulses of 10 sec each at 35% amplitude followed by a 40 sec rest period on ice between each pulse. Sheared DNA fragments (ranged in size from 200-600 bps as assessed by agarose gel electrophoresis) were immunoprecipitated using the 5hmC rabbit polyclonal antibody from the kit. qPCR was performed in a 25 µl reaction containing 2.5 µl of the eluted DNA using iTAC SYBGreen in a Bio-Rad iCycler. The relative enrichments (after normalization against control IgG) of the indicated DNA regions were calculated using the Percent Input Method according to the manufacturer's instructions.

Plasma insulin, leptin, and corticosterone
For insulin and leptin, blood samples were collected in EDTA tubes (Microtainer with K2EDTA, BD, 365974) by cardiac puncture of terminally anesthetized animals between 9:00 and 11:00. For corticosterone, blood samples were obtained via retroorbital bleeding between 19:00 and 20:00. The tubes were centrifuged at 2,000 x g at 4 °C for 20 min, and plasma was collected and stored at -80 °C until use. Plasma insulin, leptin, and corticosterone levels were measured using Mouse Insulin ELISA kit (Crystal Chem, 90080), Mouse Leptin ELISA kit (Crystal Chem,90030), and Corticosterone ELISA kit (Enzo, ADI-900-097), respectively, according to the manufacturer's instructions.

Behavioral Tests
For all behavioral tests, mice were transferred to the testing room 1 h prior to testing for acclimation to the environment. All behavioral apparatus was wiped with 70% ethanol prior to each trial and between trials. The tail suspension test (TST) (12) and the forced swim test (FST) (13) lasted for 6 min and the total amount of immobility time was measured for each animal and considered as an index of "depressive-like" behavior. For the TST, cylindrical plastic tubes were placed at the base of the tail to prevent tail climbing.

Statistical Analysis
All statistical analyses were performed using GraphPad Prism version 8 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com) and are presented as mean ± SEM. Two-tailed Student's t tests (or as otherwise indicated) were used to compare means between groups. P < 0.05 was considered significant.

Supplemental Figures
Supplemental Figure 1